Deuterium-Stabilised Ribonucleic Acid (RNA) Molecules Displaying Increased Resistance to Thermal and Enzymatic Hydrolysis, Aqueous Compositions Comprising Stabilised RNA Molecules and Methods for Making Same

ABSTRACT

The invention relates to the field of RNA stabilisation, and more particularly to the use of deuterium oxide (D2O) during storage and/or synthesis of RNA molecules. Described herein are deuterium-stabilised ribonucleic acid (RNA) molecules that display an increased resistance to thermal and enzymatic hydrolysis. Also described are aqueous compositions comprising stabilized RNA molecules and methods for making same. The invention is particularly useful for in the manufacture of RNA-based therapeutics, such as mRNA vaccines, to render them less sensitive to temperature fluctuations.

CROSS REFERENCE TO RELATED APPLICATION

The present application claims priority to U.S. patent application Ser.No. 17/522,946 filed on Nov. 10, 2021 as well as to U.S. ProvisionalApplication Ser. No. 63/112,370 filed on Nov. 11, 2020 and U.S.Provisional Application Serial No. U.S. 63/114,418 filed on Nov. 16,2020. The entire content of U.S. patent application Ser. No. 17/522,946,U.S. Provisional Application Ser. No. 63/112,370, and U.S. ProvisionalApplication Serial No. U.S. 63/114,418 is incorporated herein byreference.

FIELD OF THE INVENTION

The invention relates to the field of RNA stabilisation, and moreparticularly to the use of deuterium oxide (D₂O) during storage and/orsynthesis of RNA molecules.

BACKGROUND OF THE INVENTION

Numerous messenger ribonucleic acid (mRNA) vaccines are being developedcurrently by various pharmaceutical companies around the world to curbthe COVID-19 pandemic. In addition, mRNA therapies are beinginvestigated in a number of medical conditions. This increase ofinterest in RNA pharmaceutics may help to pave the road for mRNAtherapeutics in numerous other fields as well.

However, RNA molecules are inherently unstable and prone to bothnon-enzymatic and enzymatic hydrolysis, which is a major issue duringprocessing, transport, and storage. One of the most critical factorsthat needs to be controlled is temperature. All the mRNA vaccines andother mRNA-based therapeutics are sensitive to temperature fluctuations,which can accelerate their degradation. Therefore, there is an urgentneed for thermostable mRNA therapeutics.

Some groups have studied a potential role for deuterium oxide (D₂O) as athermal stabilizer. For instance, the use of D₂O has been described forthermostabilizing a live attenuated oral polio vaccine (Wu R. et al.,Vaccine, Vol 13, No. 12, pp. 1058-1063 (1995); Newman J. F. E. et al.,Vaccine, Vol. 15, pp. 1431-1435 (1995); Milstien J. B et al., Journal ofInfectious Diseases (1997) doi:10.1093/infdis/175.supplement_1. s247;and Sen A. et al., Expert Review of Vaccines (2009)doi:10.1586/erv.09.105, and Pathak A. K. and Bandyopadhyay T., J. Chem.Phys. 146, 165104 (2017), doi.org/10.1063/1.4982049). However, theeffects were observed with whole attenuated polio viruses andthermostabilization of naked RNA molecules was not studied nordemonstrated. Moreover, the effects of mRNA synthesis in D₂O on the mRNAstability was never addressed and RNA tautomerism was not implicated.

The use of deuterated ribonucleotides during synthesis of RNA moleculeshas been studied as well. International PCT Patent Publication No. WO2019/158583 of Ethris GmbH discloses the use of deuterated adenosine,cytidine, guanosine, and/or uridine residues for obtainingpolyribonucleotide with reduced immunogenicity. U.S. Patent ApplicationPublication Nos. US 2015/0119665, US 2015/0252071 and US 2015/025207 toASED LLC. describe, among other things, the synthesis of deuteratednucleobases, deuterated nucleosides, deuterated oligonucleotides, anddeuterated RNAs having potential for therapeutic uses. U.S. Pat. No.5,721,350 describes the deuterated nucleotide and nucleoside units whichare used to synthesize strands of RNA and DNA in NMR applications.International PCT Patent Application Publication No. WO 1992/001673 ofMedical Research Council describes the synthesis spin labelledribonucleosides and ribonucleotides that may comprise deuterium, anduses of these compounds as probes, for example in protein structure andorientation studies. International PCT Patent Application PublicationNo. WO 2019/219070 of Chia Tai Tianqing Pharmaceutical Group Co. Ltd.describes deuterated oligonucleotides and uses thereof in treatinghepatitis B virus infection. However, these patent documents do notteach deuterium as a RNA stabilizer in aqueous solution, let aloneincreased resistance of RNA molecules to thermal or enzymaticdegradation.

Hydrogen bonds play an important role in structural integrity andfunctionality of most known biomolecules including secondary andtertiary structures of nucleic acids, secondary, tertiary, andquaternary structure of proteins and of biopolymers (Li X. Z., WalkerB., and Michaelides A. Proc. Natl. Acad. Sci. U.S.A. (2011)doi:10.1073/pnas.1016653108). Ingle et al. (Nucleic Acids Res. (2014)doi:10.1093/nar/gku934) have found that substituting deuterium forprotium at a ribose 5′-carbon produces a kinetic isotope effect oncleavage but this phenomenon appeared to be highly dependent on thenucleotide sequence of the RNA molecule. Hohlffelder et al. (Biomed Res.Int. (2013) doi:10.1155/2013/592745) demonstrated that D₂O increasestranscriptional activity of T7 RNA Pol but any potential effect onstabilization or thermal degradation of RNAs was not investigated norshown.

Therefore, there is still a need for RNA-based therapeutics thatcomprises thermostable RNA molecules resistant to temperaturefluctuations.

There is also a need for compositions comprising stabilised RNAmolecules, including mRNAs and for methods for stabilizing RNAmolecules.

There is particularly a need for methods directed at reducing thermaldegradation of a RNA molecule, wherein the RNA molecule is synthesizedin the presence of deuterium and/or wherein the RNA molecule stored inthe presence of deuterium.

The present invention addresses these needs and other needs as it willbe apparent from the review of the disclosure and description of thefeatures of the invention hereinafter.

BRIEF SUMMARY OF THE INVENTION

According to one aspect, the invention relates to an aqueous compositioncomprising stabilised ribonucleic acid (RNA) molecules, said aqueouscomposition comprising at least one of: (i) RNA molecules and deuteriumfor stabilising the RNA molecules; and (ii) deuterium-stabilised RNAmolecules that have been synthesised in the presence of deuterium.Preferably, the aqueous composition comprises both (i) deuterium insolution and (ii) stabilised RNA molecules that have been synthesised inthe presence of deuterium in solution.

Preferably the RNA molecules incorporates deuterium. For instance theRNA molecules may comprise deuterated ribonucleoside tri-phosphates(rNTPs). For instance, the RNA molecules may comprise substitution ofprotium atoms by deuterium atoms. Particularly, the RNA molecules maycomprise a deuterium atom in the 2′OH-group on the ribose sugar moiety.

The stabilised RNA molecules may display increased structural integrityof their primary and/or secondary structure, compared to non-stabilizedRNA molecules. They may also display increased resistance to degradationcompared to non-stabilised RNA molecules. Preferably, the stabilised RNAmolecules display increased resistance to at least one of (i) hydrolysisor degradation by endonucleases, and (ii) thermal degradation. Morepreferably, the RNA molecules are resistant to thermal hydrolysis (e.g.resistance for one or more days of exposure to 37° C. and/or resistanceto challenge at 45° C. or higher).

According to another aspect, the invention relates to an aqueousribonucleic acid (RNA) composition comprising at least one of: (i) afirst aqueous solution comprising deuterium-stabilised RNA molecules,said solution comprising deuterium at a concentration sufficient forstabilising the RNA molecules; and (ii) a second aqueous solutioncomprising deuterium-stabilised RNA molecules that have been synthesizedin the presence of deuterium. Preferably, the aqueous RNA compositioncomprises both (i) deuterium in solution and (ii) deuterium-stabilisedRNA molecules that have been synthesised in the presence of deuterium insolution.

Preferably, the deuterium-stabilised RNA molecules incorporatesdeuterium. For instance, the deuterium-stabilised RNA molecules maycomprise deuterated ribonucleoside tri-phosphates (rNTPs). For instance,the deuterium-stabilised RNA molecules may comprise substitution ofprotium atoms by deuterium atoms. Particularly, the deuterium-stabilisedRNA molecules may comprise a deuterium atom in the 2′OH-group on theribose sugar moiety.

The deuterium-stabilised RNA molecules may display increased structuralintegrity of their primary and/or secondary structure, compared tonon-stabilized RNA molecules. The deuterium-stabilised RNA molecules mayalso display increased resistance to degradation compared tonon-stabilised RNA molecules. Preferably, the deuterium-stabilised RNAmolecules display increased resistance to at least one of (i) hydrolysisor degradation by endonucleases, and (ii) thermal degradation. Morepreferably, the deuterium-stabilised RNA molecules are resistant tothermal hydrolysis (e.g. resistance for one day or more of exposure to37° C. and/or resistance to a challenge at 45° C. or higher).

In preferred embodiments, stabilised RNA molecules comprise messengerRNA (mRNA) molecules. Such stabilised RNA molecules may advantageouslybe components of a vaccine.

According to another aspect, the invention relates to a translationproduct obtained from translation of a mRNA molecule as defined herein.

According to another aspect, the invention relates to a translationproduct of a messenger ribonucleic acid (mRNA) molecule, wherein saidmRNA molecule consists of a deuterium-stabilised mRNA molecule, andwherein said stabilised mRNA molecule (i) has been contacted with anaqueous solution comprising deuterium and/or (ii) has been synthesisedin the presence of deuterium. In embodiments, the translation product isa protein or a polypeptide.

According to another aspect, the invention relates to a method forstabilising a ribonucleic acid (RNA) molecule. In one embodiment themethod comprises at least one of: (i) storing the RNA molecule in thepresence of deuterium; and (ii) synthesising the RNA molecule in thepresence of deuterium. In embodiments, the method compromisesconsecutive steps of: (a) synthesising said RNA molecule by forwardtranscription in an aqueous composition comprising deuterium; and (b)storing the synthesized RNA molecule of step (a) in an aqueous solutioncomprising deuterium.

In embodiments, the synthesising comprises in vitro transcription in anaqueous composition comprising deuterium. In embodiments, thesynthesising comprises incorporation of deuterium into the RNA moleculevia keto-enol tautomerization. In embodiments the synthesising comprisesin vitro transcription (e.g. forward transcription) with deuteratedribonucleoside tri-phosphates (rNTPs). According to this aspect, thepresence of deuterium reduces hydrolysis or degradation of the RNAmolecule by endonucleases. Also, deuterium reduces thermal degradationof the RNA molecule. Particularly, the presence of deuterium duringsynthesis of the RNA molecule may reduce the extent of mRNA degradationduring transcription.

According to this aspect, a stabilised RNA molecule preferably displaysan increased structural integrity of its primary and/or secondarystructure, compared to a non-stabilized RNA molecule.

According to another aspect, the invention relates to a method forreducing thermal degradation of a RNA molecule. In one embodiment, themethod comprises at least one of: (i) synthesising the RNA molecule inthe presence of deuterium; and (ii) storing the RNA molecule in thepresence of deuterium. In embodiments, the method comprises consecutivesteps of: (a) synthesising the RNA molecule by forward transcription inan aqueous composition comprising deuterium; and (b) storing thesynthesized RNA molecule of step (a) in an aqueous solution comprisingdeuterium. The synthesising may further comprise in vitro transcriptionwith deuterated ribonucleoside tri-phosphates (rNTPs).

According to this aspect, a stored RNA molecule preferably displaysreduced hydrolysis or degradation by endonucleases compared to a RNAmolecule not synthesized or stored in the presence of deuterium.According to this aspect, a stored RNA molecule displays improvedresistance to thermal degradation compared to a RNA molecule notsynthesized or stored in the presence of deuterium. According to thisaspect, a stored RNA molecule displays increased structural integrity ofits primary and/or secondary structure, compared to a non-stabilized RNAmolecule.

According to another aspect, the invention relates to a stabilised RNAmolecule obtained by any of the methods described herein.

According to another aspect, the invention relates to the use of anaqueous composition as defined herein, or use of a aqueous RNAcomposition as defined herein, and/or use of a stabilised RNA moleculeas defined herein, in the manufacture of a medicament or a vaccine.

According to another aspect, the invention relates to the use of anaqueous composition as defined herein, or use of a aqueous RNAcomposition as defined herein, and/or use of a stabilised RNA moleculeas defined herein, for immunization of a subject in need thereof.

Accordingly, another related aspect of the invention concerns animmunisation method, comprising administering to a subject in needthereof an aqueous composition as defined herein, or an aqueous RNAcomposition as defined herein, or a stabilised RNA molecule as definedherein. In embodiments, the immunization comprises injecting to thesubject a mRNA vaccine.

According to another aspect, the invention relates to a RNA-basedtherapeutic, said RNA-based therapeutic comprising thermostable RNAmolecules which are resistant to temperature fluctuations. Inembodiments, the thermostable RNA molecules comprisedeuterium-stabilised RNA molecules. In embodiments, the thermostable RNAmolecules consists of deuterium-stabilised RNA molecules.Advantageously, the thermostable RNA molecules display resistance tothermal hydrolysis (e.g. resistance for 1 day or more of exposure to 37°C., a and/or resistance a challenge at 45° C. or higher). According tothat aspect, resistance to thermal hydrolysis is greater than thermalresistance of corresponding non-stabilised RNA molecules. Inembodiments, the thermostable RNA molecules comprise messenger RNA(mRNA) molecules and the RNA-based therapeutic consists of a mRNAvaccine.

According to another aspect, the invention relates to the use deuteriumas a thermostabilizer for RNA molecules.

Additional aspects, advantages and features of the present inventionwill become more apparent upon reading of the following non-restrictivedescription of preferred embodiments, which are exemplary and should notbe interpreted as limiting the scope of the invention.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

For the invention to be readily understood, embodiments of the inventionare illustrated by way of example in the accompanying figures.

FIG. 1A depicts predicted secondary structures of mRNA moleculessynthesized using template P1, in accordance with the Examples describedherein.

FIG. 1B depicts predicted secondary structures of mRNA moleculessynthesized using template P2, in accordance with the Examples describedherein.

FIGS. 2A and 2B are pictures of an agarose gel showing electrophoresisof mRNA stored at 37° C. for 2 days in accordance with the examplesdescribed herein. mRNA was synthesized using P1 and P2 templatessynthesized and stored in light water (FIG. 2A) or synthesized andstored in D₂O (FIG. 2B), in accordance with the Examples describedherein. Description of the lanes is found in Table 1 hereinafter.

FIGS. 3A and 3B are graphs showing distribution curves used forassessing mRNA degradation, in accordance with the Examples describedherein.

FIGS. 4A and 4B are bar graphs depicting the effect of synthesis andstorage of mRNA in D₂O on mRNA preservation at 37° C. for 2, 3 or 7days, in accordance with the Examples described herein.

FIG. 5 is a dot graph depicting isotope kinetic effect in mRNA stabilityover time in H₂O and D₂O, in accordance with the Examples describedherein.

FIG. 6 is a bar graph depicting isotope kinetic effect in mRNA stabilityover time in H₂O and D₂O, in accordance with the Examples describedherein.

FIGS. 7A and 7B are pictures of an agarose gel showing electrophoresisof mRNA produced and stored in D₂O (FIG. 7A) or stored in H₂O (FIG. 7B)and subjected to 37° C. for 2 days, in accordance with the Examplesdescribed herein. Description of the lanes in found in Table 1hereinafter.

FIG. 8A is a bar graph depicting the effect of D₂O on the efficacy of T7RNA polymerase, in accordance with the examples. White bars representmRNA synthesis is in H₂O and grey bars represent _mRNA synthesis in D₂O.

FIG. 8B is a graph showing hierarchical clustering of 12 experimentalconditions, in which the performance of T7 RNA polymerase was assessed,in accordance with the Examples described herein.

FIG. 9 is a picture of an agarose gel electrophoresis of purified andnon-purified mRNAs produced in H₂O and D₂O showing increase of mRNAstabilisation during synthesis in D₂O, in accordance with the Examplesdescribed herein. Gel lanes are labelled as follows: mRNA synthesizedwith T7 RNA Pol (T7) in H₂O (H) or D₂O (D), and the mRNA was eitherpurified (p) or left non-purified (np).

FIG. 10 is a picture of an agarose gel electrophoresis of purified andnon-purified mRNAs showing that synthesis of mRNA in D₂O reduces thecontamination of mRNA with large size fragments, in accordance with theExamples described herein. Gel lanes are labelled as follows: mRNAsynthesized with T7 RNA Pol (T7) in H₂O (H) or D₂O (D), and the mRNA waseither purified (p) or left non-purified (np).

FIGS. 11A and 11B are graphs showing distribution curves used forassessing non-purified mRNA integrity during synthesis in H₂O and D₂O(FIG. 11A) or during synthesis in D₂O (FIG. 11B), in accordance with theExamples described herein. FIG. 11A: Post-IVT purification of mRNA wasomitted to compare efficiency of IVT with T7 RNA Pol in H₂O and D₂O.Solid black line represents mRNA made in H₂O and dashed line mRNA madein D₂O. A representative gel electrophoresis is shown in the insert.

FIG. 12A is a bar graph depicting statistical analysis of thedegradation zone in purified and non-purified mRNA produced in H₂O andD₂O, in accordance with the Examples described herein.

FIG. 12B is a bar graph depicting statistical analysis of a largemolecular fragment contamination zone in purified and non-purified mRNAproduced in H₂O and D₂O, in accordance with the Examples describedherein. The width of the specific signal peak at 850 bp was used as aproxy for T7 RNA Pol specificity.

FIG. 13A is a line graph depicting power analysis of the degradationzone statistics, in accordance with the Examples described herein.

FIG. 13B is a line graph depicting power analysis of large molecularweight contamination zone statistics, in accordance with the Examplesdescribed herein.

FIGS. 14A and 14B are line graphs depicting the effect of secondarystructure on mRNA stability in H₂O (FIG. 14A) or in D₂O (FIG. 14B), inaccordance with the Examples described herein.

FIG. 15A is a bar graph depicting fluorescence analysis of translationof mRNA synthesised and stored in D₂O into functional protein, inaccordance with the Examples described herein.

FIG. 15B is a picture of a western blot analysis depicting translationof mRNA synthesised and stored in D₂O into functional protein, inaccordance with the Examples described herein.

FIGS. 16A and 16B are graphs showing flow cytometry analysis of murinesplenocyte after control injection (FIG. 16A) or after injection withmRNA produced and stored in D₂O (FIG. 16A), in accordance with theExamples described herein.

FIGS. 17A, 17B and 17C are panels with pictures of a gel depictingenzymatic degradation of mRNA synthesized and stored in either D₂O orH₂O, and concentration of RNAse A dependent effect on mRNA preservationwhen it is synthesised and stored in H₂O or D₂O, in accordance with theExamples described herein.

FIG. 18 show the chemical structure of a uridine molecule.

FIG. 19 is a panel showing isolation and characterization of total RNAfrom murine primary splenocytes.

FIG. 20 is a diagram illustrating selected examples of deuteriumincorporation into mRNA molecules during mRNA synthesis via keto-enoltautomerization mechanism.

Further details of the invention and its advantages will be apparentfrom the detailed description included below.

DETAILED DESCRIPTION OF EMBODIMENTS

In the following description of the embodiments, references to theaccompanying figures are illustrations of an example by which theinvention may be practiced. It will be understood that other embodimentsmay be made without departing from the scope of the invention disclosed.Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art, to which the invention belongs.

General Overview

As is known, the most common cause of mRNA degradation duringmanufacturing and storage of RNA-based pharmaceuticals is thermalhydrolysis. The present invention addresses this problem, and otherproblems related to mRNA degradation and stability, by providingdeuterium-stabilised RNA molecules, compositions comprising stabilisedRNAs, methods for stabilising RNAs, methods for reducing thermaldegradation of RNAs, and RNA-based therapeutics comprising such RNAmolecules.

Particularly, the present disclosure describes how deuterium can be usedin synthesis and/or storage to stabilize RNA molecules, including butnot limited to clinically important RNA molecules including messengerRNA, such as mRNA within mRNA vaccines, or other RNA-based therapeutics.

As used herein, the term “deuterium” refers to a stable isotope ofhydrogen or “heavy hydrogen” (i.e. ²H or D), rather than the commonhydrogen-lisotope (¹H or H, also called protium) that makes up most ofthe hydrogen in ambient water (H₂O). As used herein, the term“deuterium” or deuterium oxide encompass related terms and moleculessuch as “deuterium oxide”, “²H₂O” and “D₂O”.

Stabilised RNAs and Compositions

One particular aspect of the invention concerns deuterium-stabilisedribonucleic acid (RNA) molecules and aqueous compositions comprisingsame.

Advantageously, a deuterium-stabilised RNA according to an embodiment ofthe invention displays an increased resistance to degradation comparedto a corresponding non-stabilised RNA molecule (i.e. a RNA moleculehaving same sequence or structure). The increased resistance may includeresistance to (i) hydrolysis or degradation by endonucleases (e.g.RNAse), and/or resistance to (ii) thermal degradation.

In embodiments of the invention, a deuterium-stabilised RNA displaysincreased resistance to thermal hydrolysis after 1 day, or 2 days, or 3days, or 4 days, or 5 days, or 6 days, or 7 days or more of exposure to37° C., compared to a non-stabilised RNA molecule.

In embodiments of the invention, a deuterium-stabilised RNA displaysincreased structural integrity of its primary and/or secondarystructure, compared to a corresponding non-stabilized RNA molecule.

In embodiments of the invention, a deuterium-stabilised RNA incorporatesdeuterium.

As used herein, the term “incorporate” or “incorporation” refers topresence of deuterium into the molecular structure of the molecule, andit encompasses integration of the deuterium isotope to the molecule viacovalent, hydrogen or other type of bonding or molecular interaction.

In embodiments of the invention, a deuterium-stabilised RNA formstighter secondary structure in D₂O protecting 2′ hydroxyl on the ribosefrom participating in nucleophilic attack on the phosphodiester bond. Inembodiments, the deuterium-stabilised RNA comprises deuteratedribonucleoside tri-phosphates (rNTPs) or utilises the D₂O solvent effector combination of both.

In embodiments of the invention, a deuterium-stabilised RNA comprisesone more deuterium atoms instead of corresponding protium atom(s). Inembodiments one or more protium atoms have been replaced by one or moredeuterium atoms (e.g. substitution or any other mechanism by which mRNAinteracts with D₂O in covalent or non-covalent fashion reducing theextent of thermal or enzymatic hydrolysis).

FIG. 18 depicts possible sites for deuteration in accordance with thepresent invention. For instance, in a uridine molecule (#110A),substitution of protium to deuterium could occur at a double bond in theuracil (5-6 position #106) or at the hydroxyl on the ribose (2′position, #108).

In embodiments, the deuterium-stabilised RNA comprises a deuterium atomin the 2′OH-group on the ribose sugar moiety. In embodiments, thedeuterium-stabilised RNA comprise a deuterium isotope in the uracilitself.

In embodiments, the deuterium-stabilised RNA comprises deuterium atom(s)that have been incorporated in the RNA chemical structure duringsynthesis. For instance, as indicated hereinbefore, the RNA molecule canbe synthesized by using deuterated ribonucleoside tri-phosphates(rNTPs).

In accordance with another embodiment, deuterium atom(s) may beincorporated in the RNA molecule during RNA synthesis due to the simplepresence of D₂O in solution. In accordance with that embodiment,deuterium incorporation into RNA molecules during RNA synthesis occursvia keto-enol tautomerization. In embodiments, the solution comprisingD₂O comprises a deuterium concentration sufficient to favorthermodynamically such incorporation. In embodiments, the aqueoussolution comprises a deuterium concentration of at least 5 atom % D, orat least 10 atom % D, or at least 20 atom % D, or at least 30 atom % D,or at least 40 atom % D, or at least 50 atom % D, or at least 60 atom %D, or at least 70 atom % D, or at least 80 atom % D, or at least 85 atom% D, or at least 90 atom % D, or at least 95 atom % D, or at least 96atom % D, or at least 97 atom % D, or at least 98 atom % D, or at least99 atom % D, or at least 99.5 atom % D, or at least 99.7 atom % D, or atleast 99.9 atom % D. In embodiments, the aqueous solution comprising D₂Ocomprises deuterium at a concentration of about 5 atom % D to about 100atom % D, or about 25 atom % D to about 99.9 atom % D, or about 50 atom% D to about 99.9 atom % D, or about 75 atom % D to about 99.9 atom % D,or about 85 atom % D to about 99.9 atom % D, or about 90 atom % D toabout 99 atom % D, or about or about 99.7 atom % D.

In other embodiments, the deuterium atom(s) are incorporated in theRNA's chemical structure by contacting an already synthesized RNAmolecule (incorporating or not deuterium) with an aqueous solutioncomprising D₂O. In accordance with a particular embodiment, deuteriumincorporation into RNA molecules during RNA synthesis occurs viaketo-enol tautomerization. In embodiments, the aqueous solutioncomprising D₂O comprises a deuterium concentration sufficient to favorsuch incorporation. In embodiments, the aqueous solution comprises adeuterium concentration of at least 5 atom % D, or at least 10 atom % D,or at least 20 atom % D, or at least 30 atom % D, or at least 40 atom %D, or at least 50 atom % D, or at least 60 atom % D, or at least 70 atom% D, or at least 80 atom % D, or at least 85 atom % D, or at least 90atom % D, or at least 95 atom % D, or at least 96 atom % D, or at least97 atom % D, or at least 98 atom % D, or at least 99 atom % D, or atleast 99.5 atom % D, or at least 99.7 atom % D, or at least 99.9 atom %D. In embodiments, the aqueous solution comprising D₂O comprisesdeuterium at a concentration of about 5 atom % D to about 100 atom % D,or about 25 atom % D to about 99.9 atom % D, or about 50 atom % D toabout 99.9 atom % D, or about 75 atom % D to about 99.9 atom % D, orabout 85 atom % D to about 99.9 atom % D, or about 90 atom % D to about99 atom % D, or about or about 99.7 atom % D.

The present invention further encompasses aqueous compositions includingRNA molecules as defined herein. In embodiments, the aqueous compositionconsists of a stabilised ribonucleic acid aqueous composition comprising(i) deuterium for stabilising the RNA molecules; and/or (ii)deuterium-stabilised RNA molecules that have been synthesised in thepresence of deuterium oxide. In other embodiments, the aqueouscomposition consists of an aqueous ribonucleic acid (RNA) compositioncomprising: (i) a first aqueous solution comprising RNA molecules, thesolution comprising deuterium at a concentration sufficient forstabilising the RNA molecules; and/or (ii) a second aqueous solutioncomprising deuterium-stabilised RNA molecules that have been synthesisedin the presence of deuterium oxide. In embodiments, the aqueouscomposition consists essentially of, or alternatively comprises, astabilised ribonucleic acid aqueous composition comprising (i) deuteriumfor stabilising the RNA molecules; and/or (ii) deuterium-stabilised RNAmolecules that have been synthesized in the presence of deuterium oxide,as well as optional additional components such as RNAase inhibitor(s),enzyme(s), salts dNTPs, etc.

The present invention is not restricted to particular RNA molecules andit encompasses stabilization of various types of RNAs including, but notlimited to, total RNA, mRNA, siRNA, shRNA, etc. In embodiments, the RNAmolecule consists of a messenger RNA (mRNA) molecule. In embodiments,the mRNA molecule is a component of a therapeutic (e.g. a vaccine orelse). The RNA molecule may be obtained from different source, includingchemical synthesis, in vitro synthesis, in vivo synthesis, isolated orpurified from different sources (e.g. prokaryotic or eukaryotic cells ororganisms, viruses, etc.).

Translation Products

Another particular aspect of the invention concerns translation productsobtained from translation of a RNA (e.g. mRNA molecule) as definedherein.

In one embodiment of the invention, the translation product consists ofthe translation product of a mRNA molecule, the mRNA molecule consistingof a deuterium-stabilised mRNA molecule, whereas the stabilised mRNAmolecule: (i) has been contacted (e.g. stored) with an aqueous solutioncomprising deuterium; and/or (ii) has been synthesized in the presenceof deuterium.

In embodiments, the translation product is a protein or a polypeptide.

In embodiments, stabilised RNA molecules in accordance with theinvention can be integrated into living cells (e.g. in vitro, ex vivo,or in vivo) and they can be transcribed into functional proteins orpolypeptides.

Methods of manufacture and methods of use

Additional particular aspects of the invention concern methods formaking RNA molecules as defined herein (e.g. deuterium-stabilised RNAs),methods for methods for stabilising ribonucleic acid (RNA) molecules andmethods for reducing thermal degradation of RNA molecules.

In embodiments, the method for making deuterium-stabilised RNA moleculesas defined herein comprise synthesising the RNA molecules in an aqueousreaction media comprising D₂O. In embodiments, the method for makingdeuterium-stabilised RNA molecules as defined herein comprisessynthesising the RNA molecules by using deuterated ribonucleosidetri-phosphates (rNTPs).

In embodiments, the method for stabilising a ribonucleic acid (RNA)molecule comprises at least one of: (i) storing the RNA molecule in thepresence of deuterium; and (ii) synthesising the RNA molecule in thepresence of deuterium.

In embodiments, the method for reducing thermal degradation of a RNAmolecule, comprises at least one of: (i) synthesising the RNA moleculein the presence of deuterium; and (ii) storing the RNA molecule in thepresence of deuterium.

In embodiments, and as explained hereinbefore, in accordance with thesemethods, deuterium atoms may be incorporated in the RNA molecule duringRNA synthesis, and/or after RNA synthesis, due to the simple presence ofD₂O in solution, for instance but not limited to via keto-enoltautomerization of the RNA molecule.

In embodiments the RNA synthesis is carried out by in vitrotranscription (e.g. forward transcription) in an aqueous compositioncomprising deuterium.

In embodiments these methods comprise at least two consecutive steps of:(a) synthesising the RNA molecule by forward transcription (e.g. invitro transcription) in an aqueous composition comprising deuterium; and(b) storing the synthesized RNA molecule of step (a) in an aqueoussolution comprising deuterium. In embodiments, the synthesising stepcomprises forward transcription (e.g. in vitro transcription) withdeuterated ribonucleoside tri-phosphates (rNTPs).

In accordance with these methods, the presence of deuterium in thereaction media and/or in the RNA storage media provides one or more ofthe following benefits:

-   -   i. reduction of hydrolysis or degradation of the RNA molecule by        endonucleases (e.g. reduce affinity of RNA to endonucleases or        else)    -   ii. reduction of thermal degradation (e.g. hydrolysis) of the        RNA molecule, for instance reduction of degradation over 0° C.        such as at 0-45° C., or at 37° C. or a challenge at 45° C. or        higher;    -   iii. reduction of mRNA degradation during transcription;    -   iv. increased structural integrity of the primary and/or        secondary structure of the deuterium-stabilised RNA molecule,        compared to a non-stabilized RNA molecule;    -   v. increased structural integrity of the tertiary and/or        quaternary structure of the deuterium-stabilised RNA molecule,        compared to a non-stabilized RNA molecule;    -   vi. increasing RNA half-life;    -   vii. increasing bioavailability of the RNA molecule for its        substrate (i.e. ribosomes, other RNA molecules, etc.).

Accordingly, the present invention further encompasses the use ofdeuterium as a thermostabilizer when used as a solvent. In embodiments,deuterium is used as a thermostabilizer for RNA (e.g. mRNA, siRNA,shRNA, etc.) and its thermostabilizing activity is particularly usefulfor reducing hydrolysis and/or degradation of RNA molecules, including,but not limited to, during extended exposures (e.g. 1 day, or 2 days, or3 days, or 4 days, or 5 days, or 6 days, or 7 days or more) to 37° C.,and/or during a challenge at 45° C., or at 50° C., or at 55° C., or at60° C., or at 65° C., or at higher temperatures. The present inventionfurther encompasses the use of deuterium for RNA stability duringrenaturation process when the temperature decreases. In embodimentsdeuterium is used as a thermostabilizer for enzymes and/or for enzymaticactivity.

Therapeutical Applications

The RNA molecules in accordance with embodiments of the presentinvention may find numerous applications as research tools andtherapeutics (e.g. RNA chemistry, nanofabrication, delivery systems,immunization, etc.).

Potential therapeutic applications of the RNA molecules of the inventioninclude, but are not limited to, immunization against pathogens, cancerimmunotherapies, infectious disease vaccines, allergy tolerization,protein-replacement and supplementation therapies, genome engineeringand genetic reprogramming.

Accordingly, an additional aspect of the invention concerns RNA-basedtherapeutics comprising aqueous ribonucleic acid (RNA) compositions asdefined herein and/or comprising stabilised RNA molecules as definedherein (e.g. mRNA, siRNA, shRNA, etc.). In one embodiment, the RNA-basedtherapeutic comprises thermostable RNA molecules resistant totemperature fluctuations. In embodiments, the thermostable RNA moleculesdisplay resistance to thermal hydrolysis after 1 day, or 2 days, or 3days, or 4 days, or 5 days, or 6 days, or 7 days or more of exposure to37° C. In embodiments, the thermostable RNA molecules display resistanceto thermal hydrolysis after a challenge at 45° C., or at 50° C., or at55° C., or at 60° C., at 65° C. In embodiments, the above resistance tothermal hydrolysis is greater than thermal resistance of correspondingnon-stabilised RNA molecules. In embodiments, the RNA molecule consistsof a messenger RNA (mRNA) molecule.

In embodiments, aqueous compositions and/or stabilised RNA molecules asdefined herein are used in the manufacture of a therapeutical product(e.g. a medicament, an active pharmaceutical ingredient and/or avaccine) and/or for research purposes. In embodiments, aqueouscompositions as defined herein and/or stabilised RNA molecules asdefined herein are for administration to a subject in need thereof (e.g.for injection of the RNA to the subject). The term “subject” includesmammals in which administration of RNA molecules is desirable. The term“subject” includes domestic animals (e.g. cats, dogs, horses, pigs,cows, goats, sheep), rodents (e.g. mice or rats), rabbits, squirrels,bears, primates (e.g., chimpanzees, monkeys, gorillas, and humans), wildanimals such as those living in zoos (e.g. lion, tiger, elephant, andthe like), and transgenic species thereof. Preferably, the subject is ahuman, more preferably a human patient in need of treatment.

In embodiments, aqueous compositions as defined herein and/or stabilisedRNA molecules as defined herein are used for immunization and/or forother therapeutic-related intervention(s) of a subject in need thereof(e.g. for injection of the RNA to the subject).

In embodiments, the vaccine is a mRNA vaccine. In embodiments, thevaccine is for immunization against a viral or other pathogen. Inembodiments, the vaccine is a vaccine against Covid-19.

Those skilled in the art will recognize, or be able to ascertain, usingno more than routine experimentation, numerous equivalents to thespecific procedures, embodiments, claims, and examples described herein.Such equivalents are considered to be within the scope of thisinvention, and covered by the claims appended hereto. The invention isfurther illustrated by the following examples, which should not beconstrued as further or specifically limiting.

EXAMPLES

This section provides examples set out to evaluate the effects of mRNAsecondary structure, rNTPs deuteration and mRNA synthesis in deuteratedenvironment on the subsequent mRNA stability to thermal hydrolysis andfunctionality in vitro and in vivo.

The present examples demonstrate, among other things, that synthesis andstorage of mRNA in deuterium oxide improves mRNA resistance to thermaland enzymatic hydrolysis. Particularly, the present examples focus onthe effect of synthesis and storage of mRNA in D₂O on the mRNA stabilityduring different temperature challenges. To the best of the inventors'knowledge, this is the first study to address the mRNA stabilization bydeuterium oxide in comprehensive way.

Materials and Methods

Plasmids

Two plasmids with pCDNA3.1 backbone were engineered to contain sequencesthat can be transcribed to make mRNA which is coded to make GreenFluorescent Protein (GFP). The GFP mRNA sequences that differ in theirGC content, but code for the same amino acids. The two templates wereused to test the hypothesis that different GC content will affect mRNAstability in D₂O due to the differences in the mRNA secondary structure.The constructs contained CMV-T7-SP6-GFP regions. The GFP RNA derivedfrom plasmid 1/P1 had GC % of 38.8% and the GFP RNA derived from plasmid2/P2 had GC % of about 62.2%.

The GFP regions of the plasmids were analysed for secondary structuresusing the prediction tool RNAfold WebServer™ form University of Vienna.The minimum free energy (MFE) of P1 was −141.30 kcal/mol and P2 was−256.3 kcal/mol.

Bacterial Transformation and Culture

Competent E. coli cells (Fisher Scientific OMNIMAX2™ #C854003) andplasmid DNA mixture was incubated on ice for 20-30 mins and thereaftersubjected to heat shock transformation by being placed in a 42° C. waterbath for 45 secs and on ice for 2 minutes. Luria Broth™ (LB) media (EMDCat #1.10285.0500) without antibiotic was added to the bacteria andgrown in 37° C. shaking incubator for 45 min at 350 rpm. The transformedcells were plated on LB agar (EMD Cat #1.10283.0500) containingAmpicillin (50 ug/mL) and incubated at 37° C. overnight.

From the LB agar plate, 3-4 colonies were picked and inoculated intoliquid LB media containing Ampicillin at 50 ug/mL and incubated at 37°C. for 12-18 hr in a shaking incubator at 350 rpm.

A small amount of the overnight culture was added to 50% glycerol(Fisher Scientific™ Cat #M-12585) in a cryovial and frozen at −80° C.for future use.

Plasmid DNA Isolation

500 mL of the overnight culture was used for the plasmid purification byQIAGEN™ Plasmid Maxi kit (Cat #12161) as described by the manufacturer.In this method, bacterial lysates were cleared by centrifugation. Thecleared lysate was then loaded onto the anion-exchange tip where plasmidDNA was selectively bound under appropriate low-salt and pH conditions.RNA, proteins, metabolites, and other low-molecular-weight impuritieswere removed by a medium-salt wash, and pure plasmid DNA was eluted inhigh-salt buffer. The DNA was concentrated and desalted by isopropanolprecipitation, collected by centrifugation, and resuspended in TEbuffer.

Linearization and Clean-Up of Digested Plasmid DNA

The purified DNA was quantified using Nanodrop™ spectrophotometer(ThermoFisher Scientific) and 10 ug of DNA was linearized in a 100 ulreaction volume, using restriction enzyme Xbal (New England Biolabs Cat#R0145S) with cut site: T/CTAGA. The Serial Cloner™ software, version2.6.1 was used to identify the restriction enzyme with a single cut sitein the plasmid:

5′...T^(▾)CTAGA...3′ 3′...AGATC_(▴)T...5′

The restriction digestion mixture was incubated at 37° C. for 1 hourfollowed by heat inactivation at 65° C. for 20 minutes. This was thencleaned-up using silica membrane based QIAquick™ PCR purification kitthat binds DNA in high-salt buffer and elution with low-salt buffer. Theprotocol uses a bind-wash-elute method. The linearized DNA product wasthen run on 1.5% agarose gel and the product size corresponded to 1200base pairs on the 100 bp DNA ladder (New England Biolabs Cat #N3231S).The linearized DNA was quantified using nanodrop and 1-1.5 ug was takenfor in vitro transcription.

In Vitro Transcription

Three different IVT protocols were set-up for each of the plasmid byusing either normal NTPs (New England Biolabs cat #E2040S), a mix ofpartially deuterated NTPs (Cambridge Isotope Laboratories Cat #DLM—7862)or deuterated UTP (Millipore Sigma Cat #902454-10MG) mixed with regularATP, CTP and GTP. The template DNA was mixed with nucleotides and T7 RNApolymerase mix of the HiScribe™ IVT kit (New England Biolabs, Cat#E2040S). The reaction mixture was mixed thoroughly, pulse-spun andincubated at 37° C. for 2 hours. Template DNA was removed by setting upa reaction with DNase I (Qiagen cat #79254) at 37° C. for 15 minutes.The synthesized RNA was then purified using New England Biolab'sMonarch™ RNA Cleanup Kit (Cat #T2050). For analysis of mRNA degradationand large molecular weight fragment contamination this was modified asfollows. The IVTs were performed for 4 hours at 37° C. and 400 ng ofmRNA was immediately resolved on 1.5% agarose gel. The data wereacquired with Image J and intensities of entire lanes were plotted inPython™.

The RNA was eluted into 50 uL of H₂O or D₂O. The elution in H₂O was usedto test the isotope kinetic effect exclusively while elution on D₂O wasused to evaluate combined solvent and isotope kinetic effects. Effect ofmRNA synthesis in fully deuterated environment on the mRNA stability wastested by synthesising mRNA and storing in D₂O followed by thermalhydrolysis.

Thermal Degradation Test

RNA aliquots (400 ng) were subjected to 45° C. and 65° C. for 10 min, 60min and 18 hours in a thermocycler. Additionally, the mRNAs weresubjected to the 37° C. treatment for 2, 3, 5, and 7 days. RNA wasresolved on 1.5% agarose gel stained with ethidium bromide forvisualization. Markers such as 100 bp DNA ladder (New England BiolabsCat #N3231S) and ssRNA ladder (New England Biolabs Cat #N0362S) wereused for molecular size estimation. The effect of the mRNA secondarystructure on the stabilization by D₂O measured as degree ofpreservation.

RNA Integrity Analyses

ImageJ™ software was used for quantification of the RNA signal formagarose gel images. A rectangular region of interest was positioned tocover a maximum amount of signal in each lane. Without altering theregion of interest, pixel values for each lane were recorded. Python™programming was used for further analysis and interpretation, using acustom algorithm: the area under the signal curve (AUC) normalized tothe signal mean was used to estimate the intensity of the signal at theexpected 850 bp region and in the degradation zone. The shift of thesignal intensity from the 850 bp to the degradation zone was denoted aspeak shift. In the gels where a signal shift was not detected, the valueof the shift was denoted as 1. Degree of RNA preservation was defined asDeg_P=AUC_(Exp)×Mean_(Exp)/AUC_(Control)×Mean_(Control).

In an experiment evaluating the effect of synthesis in D₂O on mRNAintegrity and T7 Pol specificity, the area under the curve correspondingto the degradation zone (below the template-specific signal) was used.The beginning of the degradation zone was arbitrarily set at one-thirdof the peak height, where the spread of the curve started to becomeprominent. Due to the notable background signal in the high molecularweight contamination zone (above the template-specific signal), we choseto use the width of the template-specific signal peak at ⅓ of the peakheight (i.e. after degradation zone) to denote the peak width. A widepeak would indicate an increased occurrence of mRNA products withdifferent molecular weights.

Furthermore, undegraded control RNA and RNA subjected to thermalhydrolysis were screened using Agilent Bioanalyzer 2100™ for RNAintegrity numbers and analysis of the smear/degradation zone.

Capping of mRNA and In Vitro Translation

The in vitro transcribed RNA was capped afterwards to facilitateinitiation of translation and translational competence for downstream invitro translation experiment. The capping was done using New EnglandBiolabs™ capping kit (Cat #M2080), which utilizes vaccinia virus cappingenzyme (VCE), GTP and the methyl donor, SAM.

The Retic Lysate IVT Kit™ from Ambion (Cat #AM1200) was used to carryout in vitro translation. Capped RNA templates (1 ug) were mixed with20×translation mix, Met amino acid and reticulocyte lysate in a 50 uLreaction volume. A no-template control was used to subtract thebackground fluorescence. The mixture was then incubated at 30° C. for 90minutes. The product was transferred to Optiplate™ (PerkinElmer cat#6005270) for reading in fluorescence plate reader (Berthold TechTristar 2™) at an excitation wavelength of 485 nm and emissionwavelength of 530 nm.

In Vivo Translation of GFP IVT mRNA

It has been demonstrated that naked (and non-deuterated) mRNA injectedinto mice results in translation of the cognate protein it codes for,this phenomenon constituting the basis for mRNA therapeutics. In thisexample, we proceeded to validate that mRNA synthesized and stored inD₂O can be translated into a functional protein when injected into mice.400 uL of in vitro transcribed mRNA (10 ug/uL) was injectedintraperitoneally into C57BL/6 mice (Jackson Laboratory-Bar Harbor, Me.,USA) to assess in vivo translation of GFP. After in vitro transcriptionin D₂O, mRNA was resuspended in D₂O at 1.2 mg/ml. C57BL6 mice oftwenty-week-old were injected interperitoneally with 0.5 ml of mRNAsolution or H₂O as a control. After 24 hrs, mice were euthanized, andtheir spleens were dissected and homogenized to obtain a single cellsuspension, which was analyzed by flow cytometry. Murine splenocyteswere prepared in cold phosphate-buffered saline (PBS) (Multicell Cat#311-010-CL). Flow cytometry data were collected (CytoFLEX™ BeckmanCoulter, Brea, Calif., USA) and analyzed using CytExpert™ (Version2.4.0.28, Beckman Coulter Inc.). FITC (Fluorescein-5-isothiocyanate)channel with an excitation peak at 491 nm and an emission peak at 516 nmwere used to analyze GFP. At least 100 000 cells were gated on the GFPpositive cells. Background fluorescence was set as 0.01% positive cellsusing control. The established gates were applied to samples frommRNA-treated animals. The result confirms a robust expression of themRNA template.

Statistical Power Analysis.

To evaluate the validity of the sample size we used power analysis withsignificance level α=0.05.

Statistical power analysis of mRNA synthesis in H₂O and D₂O experimentaldata. Power analysis only considers the scenario when true nullhypothesis is correctly rejected (true positive). It calculatesprobability of finding the difference when there is a difference betweenmeans of two populations. High probability from 0.8 to 1 suggest theability of detection of true difference. Statistical power depends oneffect size, variability of the data that depends on the number ofobservations, and confidence/significance value a. Value a defined bythe research as a cut of desirable statistical significance. The mostwidely accepted level of statistical significance accepted in the peerreview scientific publication and the court of law of majority of thecountries including USA and Canada is 0.05.

Therefore, given the pilot data, power analysis gives information aboutnumber of samples required to demonstrate statistically (at the level α)significant differences between means of two population. Also, Poweranalysis provides the information about statistical power in existingdata.

Example 1: Synthesis and Storage of mRNA in D₂O Protects mRNA fromThermal and Enzymatic Hydrolysis and Improves the TranscriptionEfficiency

Predicted secondary structures of mRNA molecules synthesized usingtemplate P1 and template P2 is shown in FIG. 1A and FIG. 1B. ForTemplate P1 MFE: −141.30 kcal/mol, GC: 38.8% and for template P2 MFE:−256.30 kcal/mol, GC: 62.3%. MFE denotes minimal free energy. P2 mRNAwith higher GC content demonstrates a more structured and stablesecondary structure as expected.

To test the effect of synthesis and storage of mRNA in D₂O on the mRNAstability during different temperature, this study focused on the effectof synthesis and storage of mRNA in D₂O on the mRNA stability duringdifferent temperature a series of molecular tools were used in differenttemperature challenge paradigms, as shown in Table 1.

TABLE 1 Molecular tools used to study the effect of D₂O on mRNAresistance to thermal hydrolysis in different temperature challengeparadigms Code name Description 1 Plasmid 1 NTP-H GC 38.8% regular rNTPssynthesis and storage in H₂O 2 Plasmid 1 dNTP-H GC 38.8% deuteratedrNTPs synthesis and storage in H₂O 3 Plasmid 1 mUTP-H GC 38.8%deuterated Uracil synthesis and storage in H₂O 7 Plasmid 1 NTP-D GC38.8% regular rNTPs synthesis and storage in D₂O 8 Plasmid 1 dNTP-D GC38.8% deuterated rNTPs synthesis and storage in D₂O 9 Plasmid 1 mUTP-DGC 38.8% deuterated Uracil synthesis and storage in D₂O 4 Plasmid 2NTP-H GC 62.3%% regular rNTPs synthesis and storage in H₂O 5 Plasmid 2dNTP-H GC 62.3% deuterated rNTPs synthesis and storage in H₂O 6 Plasmid2 mUTP-H GC 62.3% deuterated Uracil synthesis and storage in H₂O 10Plasmid 2 NTP-D GC 62.3% regular rNTPs synthesis and storage in D₂O 11Plasmid 2 dNTP-D GC 62.3% deuterated rNTPs synthesis and storage in D₂O12 Plasmid 2 mUTP-D GC 62.3% deuterated rNTPs synthesis and storage inD₂O

Results of the thermal degradations tests are shown in FIGS. 2A and 2B.The decrease in template-specific signal (850 bp) in the mRNAs exposedto 37° C. for 48 hours that were produced and stored in the light wateris prominent (FIG. 2A). In the mRNAs that were produced and stored inD₂O this trend was not observed (FIG. 2B).

We denote the diffused signal below 850 bp as degradation zone becausethe products of mRNA degradation are of smaller molecular weight andtravel farther in the gel. The degradation zone signal was higher in themRNAs that were produced and stored in light water than in the mRNAsthat were produced and stored in D₂O.

Note that there are mRNA fragments of size larger than 850 bp. They aredetected as diffuse signal of molecular weight greater than the specificsignal (850 bp in this study) This effect is commonly observed when T7RNA polymerase is used and are attributed to the 3′ transcriptextension. There were fewer 3′ extension products in mRNA that wereproduced and stored in D₂O.

From the data shown on FIGS. 2A-2B, it appears that D₂O has a dualeffect on mRNA: 1) it protects it from the thermal hydrolysis,demonstrated as fewer small fragments and 2) improves the specificity ofT7 RNA Pol, demonstrated as fewer higher molecular weight products. Toquantify and statistically analyze the data obtained from the gelelectrophoresis experiments we designed the approach illustrated in FIG.3 .

A strategy was designed for assessing mRNA degradation. mRNAs wereresolved using 1.5% agarose gels and stained with Et-Br as described inMaterials and Methods section. The data were acquired using Image J™ andprocessed with Python™ using proprietary script. Briefly, mRNA istraveling from the negative to positive terminal of the gelelectrophoresis system. As it travels it gets distributed by size. Thelargest and the heaviest molecules travel slow, and the small onestravel fast. Because the gel is stained with Et-Br, mRNA would fluoresceunder the UV light. The intensity of fluorescence is used as proxy formRNA abundance. Therefore, the sum of fluorescent signals in the gel canbe represented as distribution (FIGS. 3A and 3B).

The height of the peak of the signal intensity distribution was denotedas signal magnitude. The mean of the signal magnitude was denoted assignal mean. The area under the signal curve (AUC) normalized to thesignal mean was used to estimate the intensity of the signal at the 850bp and in the degradation zone. The shift of the signal intensity fromthe 850 bp to the degradation zone was denoted as peak shift (FIG. 3A).In the gels where signal shift was not detected the value of the shiftwas denoted as 1 (FIG. 3B). The degree of RNA preservation was definedas Deg_P=AUC_(Exp)×Mean_(Exp)/AUC_(Control)×Mean_(Control).

For the mRNA stability over time experiments mRNAs were synthesised andstored in H₂O and D₂O and then incubated at 37° C. for 2, 3, or 7 daysto extend the duration of the experiment. The mRNAs were resolved using1.5% agarose gel electrophoresis. The results of these experiments aredepicted in FIGS. 4A and 4B.

The data represented in FIGS. 4A and 4B implies a significant, potentsolvent effect of D₂O on the mRNA stabilization at 3 and 7 days ofincubation at 37° C. FIG. 4A demonstrates a significantly higher degreeof mRNA preservation (p<0.0001; Two-way ANOVA followed by Tukey test formultiple comparisons; solvent by time interaction p<0.001, Two-wayANOVA) and FIG. 4B confirms that the template-specific maximum signalshifts significantly only when H₂O was used as a solvent (p<0.05;Two-way ANOVA and Tukey correction for multiple comparisons).

The experiments in this example demonstrated a great increase of mRNAstability if mRNA was produced and stored in deuterium oxide, over theperiod of 2 days, 3 days, and 7 days exposure to 37° C. Messenger RNAmolecules the were produced and stored in the deuterium oxide showedmore than 80% preservation during 7 days storage at 37° C. Productionand storage of mRNA in deuterium oxide increased its s stability morethan 10 fold during the 7 day incubation at 37° C.

Without wishing to be bound by theory, we propose the explanationwhereby mRNA forms tighter secondary structure in D₂O protecting 2′hydroxyl on the ribose from participating in nucleophilic attack on thephosphodiester bond. This effect may at least in part be due to thesmaller size of D₂O in comparison with H₂O and due to the weakerintermolecular bonds in D₂O in comparison to H₂O.

Example 2: Isotope Kinetic Effect Resulting from mRNA Synthesis withDeuterated Nucleotides

This example aimed to understand the contribution of isotope kineticeffect on mRNA stabilization from the quantified data by comparing themRNA preservation between groups of different nucleotides used in theIVT. The isotopic kinetic effect is referred to when the variance in thedata is driven by protium to deuterium substitution in the molecule ofinterest.

In this study, rNTPs with various extent of deuteration (Table 1) wereused to introduce deuterium atoms into mRNA molecules. The IVTs wereconducted with these rNTPs in H₂O or D₂O. The isotope kinetic effect isreferred to when the variance in the data is driven by protium todeuterium substitution in the molecule of interest. The isotope kineticeffect was masked in the deuterated environment (FIG. 5 , D₂O group) butwas visible when mRNA was synthesized and stored in H₂O (FIG. 5 , H₂O).The stabilizing effect of deuterated Uracil became apparent at 3 days of37° C. exposure and reached statistical significance at one week (FIG. 6, p<0.05, 2-way ANOVA followed by Tukey test for multiple comparisons).

Until this point in the study, mRNA was either made and stored in H₂O ormade and stored in D₂O. The importance of mRNA synthesis in D₂O alonewas not addressed. To address the effect of mRNA synthesis in D₂O onmRNA stability, IVT was performed either in H₂O followed by storage ofmRNA in D₂O or in D₂O and stored in H₂O and the mRNA stability wasassessed by thermal hydrolysis experiments. FIGS. 7A and 7B showrepresentative agarose gels of the experiment. mRNA synthesized andstored in D₂O was resistant to thermal hydrolysis after 3 days of theexposure to 37° C. regardless of the template GC content or rNTPsdeuteration (FIG. 7A). However, if mRNA was produced in H₂O, thestabilization effect of D₂O was greatly reduced (FIG. 7B).

To elucidate the mechanism of mRNA stabilization during IVT, theperformance of T7 RNA pol was assayed. As shown in FIG. 8A, using fullydeuterated environment for mRNA synthesis reduced the extent of mRNAdegradation during IVT process. There were no statistical differencesbetween the mRNA quantities produced in D₂O and H₂O (One-way ANOVA,p<0.05).

We performed hierarchical clustering of the 12 conditions (FIG. 8A). TheH₂O and D₂O experimental conditions clustered together The conditionthat clustered away from all others were when mUTPs were used and theconcentration of mRNA was reduced H₂O and D₂O conditioned clusteredtogether as well.

To explore further the stabilizing effect of mRNA synthesis in D₂O, theIVTs were conducted in H₂O and D₂O and the results were compared beforeand after on-column purification of the RNA products. We hypothesizedthat on-column purification could potentially mask the true occurrenceof high and low molecular size impurities during IVT. The IVT wasperformed in H₂O and D₂O at 37° C., which is a temperature supportive ofthermal hydrolysis. FIG. 9 shows a representative agarose gelelectrophoresis of purified and non-purified mRNAs produced in H₂O andD₂O. mRNA synthesized in D₂O showed increased stability during IVT. Thespecific signal bands are sharper and more “crisp” when mRNA wasproduced in D₂O regardless of the purification status of the sample.This supports the present invention in the fact that synthesis of mRNAin D₂O reduces the degradation already during the IVT proper, asevidenced by a decreased signal from small fragments in the degradationzone (area below the specific signal).

In summary, this study used two types of deuterated rNTPs to synthasemRNA: deuterated uracil with other RNTPs not changed and a mix ofdeuterated RNTPs. The stabilization effect was observed in H₂O morereadily than in D₂O. Without wishing to be bound by any theory, wespeculate that because the preservation of mRNA in the D₂O was alreadybetween 80% and 90% the effect of substitution of non-deuterated RNTPswith the deuterated ones could be masked. In contrast, in H₂O, where thepreservation was lower the effect of deuteration of the RNTPs wasstatistically significant improvement of mRNA stability.

Example 3: Synthesis of mRNA in D₂O Reduces the Contamination of mRNAwith Large Size Fragments

We demonstrated in FIG. 8A that T7 RNA Pol performs at least as well inD₂O as in H₂O. We next studied whether synthesis of mRNA in D₂O couldreduce the contamination of mRNA with large size fragments. mRNAs weresynthesized as described and purified (p) as described in Materials andMethods or left unpurified (np). As shown in FIG. 10 , there was lesssignal intensity observed in the higher molecular weight area in mRNAsynthesized in D₂O.

The signal quantification and comparison of non-purified products aredescribed in FIGS. 11A-B and 12A-B. mRNA was subject to more degradationduring the IVT in H₂O than in D₂O, as indicated by the higher signalarising from the degradation zone (FIGS. 11A, 12A). Also, post-IVTpurification significantly (p<0.0001, Two-way ANOVA followed by Tukeycorrection) reduced the degradation zone signal in the mRNA synthesizedin H₂O (FIG. 12A) but had no effect on the quality of mRNA produced inD₂O (FIGS. 11B, 12A). The pre-purification quality of mRNA synthesizedin D₂O was comparable to purified mRNA synthesized in H₂O (FIG. 12 ).

The width of the specific signal peak at 850 bp was used as a proxy forT7 RNA Pol specificity. The narrower the peak, the lesser the spread ofmolecular weight around the expected band at 850 bp. A wide peakindicates occurrence of arbitrary elongation and degradation products.Post IVT purification significantly (p<0.001, Two-way ANOVA followed byTukey test) reduced the peak width of mRNA produced in H₂O (FIG. 12B).There was no statistically significant difference between purified andnon-purified mRNA produced in D₂O (FIGS. 11B, 12B). Generally, mRNAsynthesized in D₂O demonstrated a more compact size distribution and thebulk signal appeared at a slightly lower molecular weight (FIG. 11A,double arrow; FIG. 12B), likely due to a tighter secondary structure ofmRNA forming in D₂O. It is possible that fewer large molecular weightcontaminants occur during IVT in D₂O due to constrains that D₂O imposeson mRNA structure and T7 RNA Pol folding. Curiously, the IVT with T7 inD₂O also seems to be more efficient since it produced more of thespecific 850 bp mRNA with less template (blue arrow in FIG. 11A).

Therefore, there is less degradation and high molecular weight artifactsin non-purified mRNA synthesized in D₂O than in H₂O (FIG. 11A). Also,there is no significant effect of post IVT purification on thedegradation zone signal and high molecular weight contamination in mRNAsynthesized in D₂O as evidenced by the overlapping signal curves ofpurified and non-purified mRNA (FIG. 11B). D₂O improves mRNA stabilityand reduces contamination with large molecular weight mRNA productsduring IVT.

The results in differences in mRNA integrity after synthesis in H₂O orD₂O are also very persuasive. mRNA showed more degradation during theIVT in H₂O than in D₂O as was shown by the larger degradation zone (FIG.12A). Purification significantly reduced the degradation zone signal inmRNA synthesized in H₂O but not in D₂O (Two-way ANOVA followed by Tukeycorrection p<0.0001).

As shown in FIG. 12B, post IVT purification significantly reduced thepeak width in mRNA produced in light water, but no statisticallysignificant change was observed between purified and non-purified mRNAproduced in D₂O (Two-way ANOVA followed by Tukey correction p<0.001).

Statistical power analysis of mRNA synthesis in H₂O and D₂O experimentaldata. Power analysis only considers scenario when true null hypothesisis correctly rejected (true positive). It calculates the probability offinding the difference when there is a difference between means of twopopulations. High probability from 0.8 to 1 suggest the ability ofdetection of true difference. Statistical power depends on effect size,variability of the data that depends on the number of observations, andconfidence/significance value a. Value a defined by the research as acut of desirable statistical significance. The most widely acceptedlevel of statistical significance accepted in the peer review scientificpublication and the court of law of majority of the countries includingUSA and Canada is 0.05. In FIG. 13A the line with the triangle symbolsdepicts the increase in statistical power (y axis) as the sample sizeincreases (Number of Observations, x axis) at significance level α=0.05and the effect size of the degradation zone (AUC) is 3.38. With thiseffect size the power at n=3 is higher than 0.8 The line without symbolis given as an example of an effect size of 2. The steeper the curve thesmaller number of observations is required to achieve a high power. InFIG. 13B the line with the triangle symbols depicts the increase instatistical power (y axis) as the sample size increases (Number ofObservations, x axis) at significance level α=0.01 and the effect sizeof signal spread is 4.37. With this effect size the power at n=3 ishigher than 0.9. The line without symbol is given as an example of aneffect size of 2. The power analysis suggests that with such prominenteffect sizes 3 observations are sufficient to demonstrate significantdifferences between the groups.

Therefore, given the pilot data, power analysis gives information aboutthe number of samples required to demonstrate statistically (at thelevel α) significant differences between means of two population. Also,power analysis provides the information about statistical power inexisting data.

Power analysis is advantageous in that it provides information aboutmagnitude of the experimental effect and required sample size, whilestrict testing of null hypothesis using the threshold p value providesbinary output: reject or accept.

As is known, during manufacturing the yields of mRNA are significantlyreduced during the IVT process. The present examples demonstrate thatconducting mRNA synthesis in D₂O and storing mRNA in D₂O improved themRNA stability in two aspects. Firstly, mRNA showed less degradationduring the IVT process. This could potentially lead to better economiccharacteristics of the production process. Second, the contaminationwith large molecular-weight fragments was reduced when mRNA was made inD₂O. This could also increase the efficiency of mRNA synthesis. Withoutwishing to be bound by any theory, we propose that during mRNAsynthesis, due to tautomerism, Deuterium incorporates into mRNA backboneenhancing it's stability and resistance to thermal and enzymatichydrolysis. In this regard, FIG. 20 depicts non-exhaustive examples ofdeuterium incorporation into mRNA molecules during mRNA synthesis viaketo-enol tautomerization.

Example 4: Effect of mRNA Secondary Structure and/or GC Content onStabilization by D₂O

This experiment was designed to explore the stabilizing effect of D₂O onmRNA by focusing on analyzing the contribution of mRNA secondarystructure to the phenomenon. This was done using template plasmid P1(here “a”) which has 38.8% GC content and MFE of −141.30 kcal/mol (lessstructured and stable secondary structure), whereas template plasmid P2(here “g”) has 63.2% GC content with a MFE of −256.30 (more structuredand stable secondary structure).

The mRNA was synthesized using both templates in H₂O and D₂O andsubjected to high temperature treatment. The treatment paradigmsincluded a challenge in 45° C. (non-denaturing condition at which themRNA secondary structure is believed to be preserved) and a challenge in65° C. (denaturing condition at which the secondary structure of mRNAceases to exist). These approaches were designed to assay the effect ofD₂O stabilization on the mRNA with differing GC content and secondarystructures.

As expected, in H₂O the GC rich mRNA showed the highest stability at 45°C., measured as degree of preservation (FIG. 14A). This phenomenondisappeared at 65° C., when secondary structure no longer protectsagainst denaturation. In D₂O, the GC-rich template again demonstratedthe best stability, but stability of the GC-poor mRNA was greatlyimproved to the level of the GC-rich template in H₂O. In H₂O the GC-richmRNA showed the highest stability at 45° C., likely owing to the morestable secondary structure. This effect disappeared at 65° C. when thesecondary structure is no longer protective of denaturation (FIG. 14B).

Without wishing to be bound by theory, this could be in part due to amore compact mRNA structure in D₂O. In D₂O, both mRNA templates werestabilized at 45° C., and to a lesser degree even at 65° C., at everytime point as evidenced by higher degrees of preservation than in H₂O(FIG. 14A). At 45° C. and in D₂O the GC-rich mRNA showed the beststability. The GC-poor template was stabilized at 45° C. in D₂O close tothe degree of GC-rich template at 45° C. in H₂O.

Example 5: In Vitro Translation Capacity of mRNA Synthesized in D₂O

To be a viable option for stabilization of mRNA in synthesis andstorage, D₂O should not interfere with the translation of the mRNAtemplate into protein. This was tested by comparing the in vitrotranslation of P1 GFP mRNA synthesized and stored in D₂O or H₂O (FIGS.15A and 15B).

The amount of translated GFP directly contributes to the intensity ofthe fluorescence signal in the sample. The emission intensities werelog-transformed for normalization. There was no statisticallysignificant difference between the GFP fluorescence intensities intranslation products of mRNA templates produced and stored in H₂O or D₂O(one-way ANOVA with Bonferroni correction for multiple comparisons,p>0.05). Therefore, we conclude that mRNA produced and stored in D₂O canbe translated into a functional protein (FIG. 15A).

The protein samples were resolved on the polyacrylamide gel andtransferred to PVDF membrane (Material and Methods). The membrane wasprobed with anti-GFP antibody as described in the Western blot sectionof materials and methods. In all instances 1 ug of mRNA was used for thein vitro translation experiments. The efficiency of the in vitrotranslation of mRNA prepared and stored in D₂O was at least as good asof mRNA prepared in the H₂O (FIG. 15B).

Example 6: In Vivo Translation Capacity of mRNA Synthesized in D₂O

Flow cytometry analysis of GFP expression in mouse splenocytes after anintraperitoneal injection of mRNA synthesized and stored in D₂O is shownon the FIG. 16 . It was confirmed that the injected mRNA had resulted inthe translation of the coded protein GFP. Furthermore, although notlabeled with specific markers, the side scatter (SSC, y-axis) andforward scatter (FSC, x-axis) metrics of GFP signal suggest that themajority of GFP positive cells belong to a subset of dendritic cells.Other cell types that express GFP potentially include activatedmacrophages and neutrophils. To further phenotype GFP positive cellsmarkers such as CD11c, CD11b, MHCII, CD80, CD86, Ly6C, Ly6G can be used.

Example 7: Synthesis and Storage of mRNA in D₂O Protects it fromEnzymatic Hydrolysis

As is known, RNAse A catalyzes mRNA hydrolysis. In this study 0.01 pg ofRNAse A was used to treat 500 ng on mRNA. The mRNA then was resolved onthe agarose gel and the data were acquired with Image J™. As shown in inFIG. 17A, The degradation of mRNA was much less pronounced in mRNA thatwas synthesized and stored in D₂O than in mRNA that was produced andstored in H₂O. FIGS. 17B and 17C demonstrate concentration dependenthydrolysis by RNAse A of mRNA produced and stored in H₂O and D₂O. mRNAproduced and stored in D₂O demonstrated stronger resistance to RNAsemediated hydrolysis in concentration dependent manner.

Example 8: Isolation and Characterization of Total RNA from MurinePrimary Splenocytes

Total RNA stabilization as well as in vitro RNA transcription protocolwas tested. Total RNA was extracted and the RNA was eluted into 100 μLof D₂O or 100 uL of H₂O and quantified using Nanodrop™spectrophotometer. The experiments were designed to test the hypothesisthat replacing U with dU increases mRNAnIrx1 stability, is synthesizedde novo using T7 or SP6 RNA polymerases from existing plasmids usingcommercially available kits (Promega #E2040S HI Scribe. The Uridine inthe RNA synthesis was replaced with Uridine-D₁₃ (Sigma 902454-1MG). RNAaliquots of 30 μL (100 ng) were used for temperature degradation tests.RNA aliquots were subjected to different temperature treatments fordifferent incubation times: 65° C., 37° C. or room temperature (RT) for10, 60 min and 12 hours. This incubation was performed in a thermocyclerto avoid evaporation as previously described. Alternatively, RNAase Atreatment was used to cleaves the 3′-end of unpaired C and U residues.The RNA was resolved on 1% agarose gel and stained with EtBr forvisualization. The data was analyzed using a gel imaging system andimage J™ software. The samples we also tested using Agilent Bioanalyzer2100™ and RNA integrity numbers will be obtained.

As shown in FIG. 19 , resuspension of total RNA in D₂O resulted inincreased RNA stability at 37 degrees Celsius.

Headings are included herein for reference and to aid in locatingcertain sections. These headings are not intended to limit the scope ofthe concepts described therein, and these concepts may haveapplicability in other sections throughout the entire specification.Thus, the present invention is not intended to be limited to theembodiments shown herein but is to be accorded the widest scopeconsistent with the principles and novel features disclosed herein.

As used herein, the terms, “comprises” and “comprising” are to beconstrued as being inclusive and open ended, and not exclusive.Specifically, when used in the specification and claims, the terms“comprises” and “comprising” and variations thereof mean the specifiedfeatures, steps or components are included. These terms are not to beinterpreted to exclude the presence of other features, steps orcomponents.

The singular forms “a”, “an” and “the” include corresponding pluralreferences unless the context clearly dictates otherwise. Thus, forexample, reference to “a RNA molecule” includes one or more of suchmolecules and reference to “the method” includes reference to equivalentsteps and methods known to those of ordinary skill in the art that couldbe modified or substituted for the methods described herein.

Unless otherwise indicated, all numbers expressing quantities ofingredients, reaction conditions, concentrations, properties, and soforth used in the specification and claims are to be understood as beingmodified in all instances by the term “about”. At the very least, eachnumerical parameter should at least be construed in light of the numberof reported significant digits and by applying ordinary roundingtechniques. Accordingly, unless indicated to the contrary, the numericalparameters set forth in the present specification and attached claimsare approximations that may vary depending upon the properties sought tobe obtained. Notwithstanding that the numerical ranges and parameterssetting forth the broad scope of the embodiments are approximations, thenumerical values set forth in the specific examples are reported asprecisely as possible. Any numerical value, however, inherently containscertain errors resulting from variations in experiments, testingmeasurements, statistical analyses and such.

It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to persons skilled in the art and areto be included within the present invention and scope of the appendedclaims.

1. A method for reducing thermal degradation of a RNA molecule,comprising the step of synthesising the RNA molecule in presence ofdeuterium to obtain a deuterium-stabilised RNA molecule incorporatingdeuterium.
 2. The method of claim 1, wherein said synthesizing comprisesin vitro transcription in an aqueous composition comprising deuterium.3. The method of claim 1, wherein said synthesizing comprisesincorporation of deuterium into the RNA molecule via keto-enoltautomerization.
 4. The method of claim 1, wherein said synthesizingcomprises in vitro transcription with deuterated ribonucleosidetriphosphates (rNTPs).
 5. The method of claim 1, wherein thedeuterium-stabilised RNA molecule incorporating deuterium comprisessubstitution of protium atoms by deuterium atoms.
 6. The method of claim1, wherein the deuterium-stabilised RNA molecule incorporating deuteriumcomprises a deuterium atom in the 2′OH-group on the ribose sugar moiety.7. The method of claim 1, further comprising the step of (ii) storingthe deuterium-stabilised RNA molecule in presence of deuterium.
 8. Themethod of claim 1, wherein said synthesising provides one or more of thefollowing benefits to the RNA molecule incorporating deuterium: i.reduction of hydrolysis or degradation of the deuterium-stabilised RNAmolecule by endonucleases; ii. reduction of thermal degradation of thedeuterium-stabilised RNA molecule; iii. reduction of mRNA degradationduring transcription; iv. increasing structural integrity of the primaryand/or secondary structure of the deuterium-stabilised RNA moleculeincorporating deuterium, compared to a non-stabilized RNA molecule; v.increasing RNA half-life; and vi. increasing bioavailability of thedeuterium-stabilised RNA molecule for its substrate.
 9. The method ofclaim 1, wherein the deuterium-stabilised RNA molecule incorporatingdeuterium displays increased resistance to thermal hydrolysis after 1day of exposure to 37° C.
 10. The method of claim 1, whereindeuterium-stabilised RNA molecule incorporating deuterium displaysincreased resistance to thermal hydrolysis after a challenge at 45° C.11. The method of claim 1, wherein said the deuterium-stabilised RNAmolecule incorporating deuterium displays resistance to thermalhydrolysis after 1 day, or 2 days, or 3 days, or 4 days, or 5 days, or 6days, or 7 days or more of exposure to 37° C.
 12. The method of claim 1,wherein the deuterium-stabilised RNA molecule incorporating deuteriumdisplays resistance to thermal hydrolysis after a challenge at 45° C.,or at 50° C., or at 55° C., or at 60° C., at 65° C.
 13. The method ofclaim 1, wherein resistance to thermal hydrolysis of thedeuterium-stabilised RNA molecule incorporating deuterium is greaterthan thermal resistance of a corresponding RNA molecule notincorporating deuterium.
 14. The method of claim 1, wherein thedeuterium-stabilised RNA molecule incorporating deuterium comprisesconsists of a messenger RNA (mRNA) molecule.
 15. The method of claim 1,wherein the deuterium-stabilised RNA molecule incorporating deuterium isa component of a vaccine.